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AMP-Activated Protein Kinase-&Alpha Citation: Cell Death and Disease (2012) 3, e357; doi:10.1038/cddis.2012.95 & 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12 www.nature.com/cddis AMP-activated protein kinase-a1 as an activating kinase of TGF-b-activated kinase 1 has a key role in inflammatory signals SY Kim1, S Jeong1, E Jung1, K-H Baik1, MH Chang1, SA Kim1, J-H Shim2, E Chun*,2 and K-Y Lee*,1 Although previous studies have proposed plausible mechanisms of the activation of transforming growth factor-b-activated kinase 1 (TAK1) in inflammatory signals, including Toll-like receptors (TLRs), its activating kinase still remains to be unclear. In the present study, we have provided evidences that AMP-activated protein kinase (AMPK)-a1 has a pivotal role for activating TAK1, and thereby regulate NF-jB-dependent gene expressions in inflammatory signaling mediated by TLR4 and TNF-a stimulation. AMPK-a1 specifically interacts with TAK1 and reciprocally regulates their kinase activities. Upon the stimulation of lipopolysaccharide, AMPK-a1-knockdown (AMPK-a1KD) or TAK1-knockdown human monocytic THP-1 cells exhibit a dramatic reduction in the TAK1 or AMPK-a1 kinase activity, respectively, and subsequent suppressions of its downstream signaling cascades, which further leads to inhibitions of NF-jB and thereby productions of proinflammatory cytokines, such as TNF-a, IL-1b, and IL-6. Importantly, the microarray analysis of AMPK-a1KD cells revealed a dramatic reduction in the NF-jB-dependent genes induced by TLR4 and TNF-a stimulation, and the observation was in significant correlation with the results of quantitative real-time PCR. Moreover, AMPK-a1KD cells are highly sensitive to the TNF-a-induced apoptosis, which is accompanied with dramatic reductions in the NF-jB-dependent and anti-apoptotic genes. As a result, our data demonstrate that AMPK-a1asan activating kinase of TAK1 has a key role in mediating inflammatory signals triggered by TLR4 and TNF-a. Cell Death and Disease (2012) 3, e357; doi:10.1038/cddis.2012.95; published online 26 July 2012 Subject Category: Cancer Metabolism Transforming growth factor-b (TGF-b)-activated kinase 1 catalytic (a) subunit and two non-catalytic regulatory subunits, (TAK1) is a member of the mitogen-activated protein kinase b (30 kDa) and g (38–63 kDa). There are several isoforms for kinase kinase family and a serine/threonine protein kinase.1–4 each of the three AMPK subunits, including a1, a2, b1, b2, g1, TAK1 functions as an upstream signaling molecule of NF-kB g2, and g3.11,12 The two AMPKa variants, a1 and a2, have and MAPKs in proinflammatory signals transduced by TNF-a, been shown to have a differential localization pattern in IL-1b, and Toll-like receptor (TLR) ligands.5,6 Binding mammalian cells, with the AMPK-a1 subunit being localized in of lipopolysaccharide (LPS) to TLR4 causes recruitment of the cytoplasm, whereas the AMPK-a2 subunit localized in the MyD88, IRAK, and TRAF6 to the receptor.7,8 The TRAF6 nucleus.13,14 In terms of cell type specificity, most of the cells catalyzes synthesis of K63-linked polyubiquitin chains that express both AMPK-a1 and AMPK-a2 isoforms, whereas serve as a scaffold to recruit the TAK1 and IkB kinase (IKK) lymphcytes only express the AMPK-a1 isoform.15,16 A complexes. Recruitment of the kinase complexes facilitates previous report has addressed the relationship between T-cell autophosphorylation of TAK1 and subsequent phosphoryla- receptor-mediated signaling and AMPK-a1 through Ca2 þ - tion of IKKb by TAK1, leading to IkB degradation and calmodulin-dependent protein kinase (CaMKK)-dependent subsequent activation of NF-kB.5,6 Despite of the plausible pathway, implying that AMPK-a1 may have specific roles in mechanisms by which TAK1 is activated,6 the existence of a immune cells.15 specific upstream kinase of TAK1 in the signaling cascades Here, we demonstrate that AMPK-a1 has a pivotal role in is still ambiguous. inducing proinflammatory signals, such as TLR4 and TNF-a, The AMP-activated protein kinase (AMPK) is a serine/ through the activation of TAK1. The knockdown of AMPK-a1 threonine protein kinase that has emerged as a master sensor results in the marked reduction of TAK1 activity, and thereby of cellular energy balance in mammalian cells.9,10 The AMPK suppressions of downstream signaling cascades and the protein exists as a heterotrimer composed of a 63-kDa expression of NF-kB-dependent genes in response to LPS 1Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea and 2Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, MA, USA *Corresponding authors: E Chun, Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, 665 Huntington Avenue, Building 1, Room 904, Boston, MA 02115, USA. Tel: 617 432 3250; Fax: 617 432 3259; E-mail: [email protected] or K-Y Lee, Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chenocheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea. Tel: +82 31 299 6225; Fax: +82 31 299 6229; E-mail: [email protected] Keywords: AMP-activated protein kinase-a1; TGF-b-activated kinase 1; NF-kB; Toll-like receptor 4; TNF-a-induced apoptosis Abbreviations: TAK1, transforming growth factor-b (TGF-b)-activated kinase 1; TLR, Toll-like receptor; IKK, IkB kinase; AMPK, AMP-activated protein kinase; LPS, lipopolysaccharide; AP-1, activator protein 1 Received 30.4.12; revised 12.6.12; accepted 20.6.12; Edited by T Brunner AMPK-a1 as an activating kinase of TAK1 SY Kim et al 2 stimulation, indicating an AMPK-a1-TAK1-NF-kB axis in the activation of AMPK.17–20 We raised a possibility of the TLR4-mediated signaling. Moreover, AMPK-a1-knockdown functional regulation between TAK1 and AMPK-a1 serine/ cells are highly sensitive to TNF-a-induced apoptosis, threonine protein kinase. We first tested the molecular supporting the functional role of AMPK-a1 associated interaction between AMPK-a1 and TAK1. The overexpressed with expressions of anti-apoptotic genes regulated by the TAK1 was significantly co-immunoprecipitated with over- TAK1-induced NF-kB activity. Thus, our results suggest that expressed AMPK-a1, and the interaction was enhanced by AMPK-a1 may be one of the upstream kinases capable LPS stimulation (Figure 2a, lane 4 versus lane 5). To identify of inducing the activation of TAK1 in proinflammatory signals. the interaction domain in each other, TAK1 or AMPK-a1 truncated mutants were constructed (Supplementary Results Figure 2), and then immunoprecipitation assay was per- formed. Overexpressed AMPK-a1 was specifically co-immu- Inhibition of AMPK-a1 activity attenuates TLR4-mediated noprecipitated with all the truncated TAK1 construct, signaling pathway. Previous reports have demonstrated indicating that AMPK-a1 interacts with TAK1 through the the functional relationship between AMPK and TAK1;17–20 N-terminus of TAK1 (Figure 2b). In addition, TAK1 specifi- however, the functional role of AMPK-a1 has not yet been cally interacted with the autoinhibitory domain of AMPK-a1, elucidated in innate signal. Therefore, we have explored this AMPK-a1 (312–392) (Figure 2c). To verify the in vitro issue in human monocytic THP-1 cells with a TLR4 agonist, interaction, we further performed endogenous immunopreci- LPS. To know whether LPS stimulation induces the activa- pication assay. Consistent with in vitro interaction, endogen- tion of AMPK-a1, because it has been known that AMPK-a1, ous TAK1 was specifically coprecipitated with endogenous but not AMPK-a2, is solely expressed in human monocytic AMPK-a1, and the interaction was significantly enhanced in cells,16 THP-1 cells were treated with LPS for different time response to LPS stimulation (Figure 2d, lane 1 versus intervals and then the activation of AMPK-a1 was evaluated lane 2). These results suggest that the autoinhibitory domain with anti-pho-AMPK antibody. The phosphorylation of AMPK-a1 interacts with the N-terminus of TAK1 of AMPK-a1 was gradually increased by LPS treatment in a (Figure 2e). We next explored whether AMPK-a1 is directly time-dependent manner (Supplementary Figure 1). To able to activate TAK1 in response to LPS stimulation. further examine whether AMPK-a1 is either a positive or To find an answer for our query, we generated knockdown negative regulator in the TLR4-mediated signaling pathway, THP-1 cells against AMPK-a1 and TAK1 protein by using leading to activate NF-kB and activator protein 1 (AP-1), siRNA-contained lentiviruses (Supplementary Figure 3 and THP-1 cells were stimulated with LPS in the presence or Figure 2f). Interestingly, phosphorylations of Thr172/174 absence of different concentrations of compound C, which is and Ser 485 of APK-a1 were markedly reduced and a pharmacological inhibitor of AMPK.21 Both NF-kB and the phosphorylation of TAK1 was dramatically reduced in KD AP-1 reporter activities induced by LPS were significantly AMPK-a1-knockdown (AMPK-a1 ) THP-1 cells treated with reduced in compound C-treated THP-1 in a dose-dependent LPS as compared with that of wt THP-1 cells (Figure 2f, KD KD manner (Figure 1a, NF-kB; Figure 1b, AP-1). Furthermore, AMPK-a1 ). Interestingly, TAK1-knockdown (TAK1 ) cells the inhibition of AMPK-a1 activity resulted in a dramatic exhibited marked reduction in phosphorylation of Thr172/174 reduction in the levels of proinflammatory cytokines, such as and Ser 485 of AMPK-a1 in response to LPS stimulation KD TNF-a, IL-6, and IL-1b, induced by LPS (Figure 1c, IL-6; (Figure 2f, TAK1 ).
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